1. Field of the Invention
This invention relates to chromatography, particularly size exclusion chromatography, and to the composition of the resolving zones used in such chromatography. It also relates to a process for performing chromatographic separation.
2. Discussion of the Prior Art
U.S. Pat. No. 3,505,785 discloses superficially porous microspheriods, having an average diameter in the range of 5 to 500 microns, which are composed of an impervious core coated with a multiplicity of monolayers of colloidal inorganic particles having an average size in the range of 0.005 to 1.0 microns. U.S. Pat. No. 3,855,172 discloses microspheriods which are porous throughout and have an average diameter in the range of 0.5 to 20 microns. They are composed of colloidal inorganic refractory particles having an average diameter in the range of 0.005 to 1.0 microns.
The use of such microspheriods in chromatography, particularly size-exclusion chromatography is well-known. In such application, porous microspheroids are used as packings for the chromatograph resolving zone and functions to separate the components of a sample based on differences in hydrodynamic size of the components. The molecular weight (MW) of the components can be calculated as a function of the hydrodynamic size. A plot of the molecular weight fraction eluted at a given retention volume (V.sub.R) for a particular packing material reveals that for a given pore volume in the packing material, certain molecular weight fractions are totally excluded because of their large size and certain molecular weight fractions are totally permeating because of their small size. Between these two extremes is a range of molecular weight fractions that will be preferentially retarded by contact with the porous particles, and materials containing these molecular weight fractions can be fractionated by that particular packing material.
The actual working relationship in size exclusion chromatography is the molecular weight calibration curve which is normally a logarithmic plot of the molecular weight versus the retention volume. Molecular weight calibration curves characteristically have a substantially linear portion so that the molecular weight of a retained fraction of a sample can be determined accurately if the retention volume for that particular molecular weight fraction occurs in the linear portion of the molecular weight calibration curve and less accurately if it occurs outside that linear range. Molecular weight calibration curves characteristically have linear portions that span approximately two decades in the log molecular weight scale for a single pore size. To obtain a calibration curve with a linear portion spanning more than two decades of the molecular weight scale, the tendency is to use a chromatographic resolving zone composed of many columns, each having different pore sizes. Specifically, as taught by "Know More About Your Polymer", a 1976 publication of Waters Associates, Milford, Mass., to expand the linear range of the molecular weight calibration curve so that materials containing a wide range of molecular weight fractions can be separated and detected, four or five columns are combined, each with molecular weight calibration curves whose linear portions overlap one another.
Unfortunately, the range of expected linear molecular weight calibration does not occur. The linear portion of the molecular weight calibration curve for the combined particles can be increased in this way but the maximum appears to be only about three decades, which often is less than the range of molecular weight found in normal sample compositions.